A structural template can be defined as the approximate path that a polypeptide chain follows in space. This definition leads immediately to the following two questions that are addressed in this proposal: First, how many different sequences fold along a specific structural template? Second, which of these isomorphic structures are actually functional? We have developed spectroscopic probes of reaction center proteins that detect structural and functional perturbations elicited by mutations. While it is impossible to test experimentally all possible unique sequences in an entire protein, these methods make it feasible to test all sequences within smaller structural templates. We will employ new experimental technologies capable of generating and screening millions of different sequences and structures. Studies at this level of sequence complexity would correspond, for example, to simultaneously testing all possible amino acid residues at four different sequence positions (complexity=204=1.6x105). Alternatively, two residues could be tested at 20 different sequence positions over a larger template (220 approximately 106). In order to evaluate such large numbers of sequences, it is highly advantageous to use optical screening methods and chromophore- bearing proteins. We have recently demonstrated that optical imaging spectroscopy can be used to analyze the absorption spectrum of bacterial colonies expressing genetically modified photosynthetic reaction center proteins. (The bacterial reaction center is the only membrane protein with a structure known to atomic resolution; it has been extensively studied by spectroscopy.) Using this new imaging technique, hundreds of colonies will be simultaneously analyzed and categorized. Since the reaction center spectrum is critically dependent on the protein environment and relative orientation of the six bound chromophores, low resolution protein structural information can be inferred. Other optical assays compatible with imaging spectroscopy will be employed to assess functionality (e.g. charge-separation). These studies will generate massive databases of sequence/structural information that will be useful in developing heuristic "rules" and hypotheses for determining structural parameters in membrane proteins. Fundamental principles discovered in this prokaryotic model system will generalize to other (health related) membrane proteins which are not as well understood or amenable to structural studies.